Composition for promoting wound healing

ABSTRACT

The present disclosure relates to a composition sustainable-releasing hydrogen sulfide for promoting wound healing and the method for preparing the same. The composition for promoting wound healing comprises a carrier and a plurality of hydrogen sulfide sustained releasing microspheres, wherein the hydrogen sulfide sustained releasing microspheres comprise a hydrophobic polymer, a surfactant and sodium hydrosulfide.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Divisional Application of the U.S.application Ser. No. 17/204,914, filed Mar. 17, 2021, which claimspriority to Taiwan Application Serial Number 109134458, filed Oct. 5,2020, all of which are herein incorporated by reference in theirentireties.

BACKGROUND Field of Invention

The present disclosure relates to a composition for promoting woundhealing and the method for preparing the same. More particularly, thepresent disclosure relates to a composition sustainable-releasinghydrogen sulfide for promoting wound healing and the method forpreparing the same.

Description of Related Art

Angiogenesis is one of the important mechanisms in wound healing.However, because of long-term high blood sugar, the patients withdiabetes are prone to vascular disease, such as, vascular sclerosis orblockage, to cause the blood flow at the wound sites poor. The poorblood circulation around the wound sites results in the reduced growthand formation of the blood vessels to delay wound heal. In addition,high blood sugar will also reduce the activity of white blood cells, andthe phagocytosis and sterilization functions will become worse. Becausethe immune function is weak, the wound is continuously infected bybacterial and will inhibit the cell proliferation. This is anotherfactor in delaying the wounds healing of patients with diabetes.

Hydrogen sulfide (H₂S) regarded as the third gaseous signaling moleculefollowing the nitric oxide (NO) and carbon monoxide (CO). It has beenproved in animal experiments that hydrogen sulfide relates to therelaxation of blood vessels, the inhibition of the proliferation ofvascular smooth muscle cells, the induction of vascular smooth musclecells apoptosis, the promotion of the proliferation of microvascularendothelial cells, anti-inflammatory and antioxidant effects. In thestate of the art, it is known that the normal cells can produce lowconcentration of gaseous hydrogen sulfide in a slow rate. However, theexternal hydrogen sulfide donor is unable to really mimic the producingrate of hydrogen sulfide in human body.

Sodium hydrosulfide (NaHS) was used as an H₂S donor since NaHS canregenerate H₂S release when contact with water. However, becausehydrosulfide ion (HS⁻) in sodium hydrosulfide reacted with the hydrogenion (H⁺) from water to release hydrogen sulfide is very fast, the fastrelease will induce high concentration of hydrogen sulfide in shorttime. High concentration of hydrogen sulfide is toxic to cells and willinduce adverse effects such as, cell apoptosis or inflammation. Lowconcentration of hydrogen sulfide will promote the wound healing, butthe cytotoxicity induced by the high concentration thereof made thehydrogen sulfide not suitable to be used in medical treatment.

In the state of the art, the wound dressing which can release gaseoushydrogen sulfide has been disclosed, but the effective concentration ofhydrogen sulfide released for healing the wound can only last for about24 hours. It is needed to frequently change the wound dressing in such acircumstance which is not good for wound healing.

Thus, it demands a composition for sustainable-releasing hydrogensulfide without inducing cytotoxicity but effectively promoting thewound healing.

SUMMARY

The present disclosure is to provide a novel composition for promotingwound healing and the method for preparing the same. The presentcomposition for promoting wound healing can sustainably release hydrogensulfide, for example, at a releasing concentration in the range of 10 to50 μM/hr in 48 hours. Thus, the present composition can effectivelypromote the wound healing without inducing any cytotoxicity.

An aspect of the present disclosure is to provide a composition forpromoting wound healing comprising a carrier and a plurality of hydrogensulfide sustained-releasing microspheres. The hydrogen sulfidesustained-releasing microspheres comprise a hydrophobic polymer, asurfactant and sodium hydrosulfide, wherein the hydrophobic polymer canbe, for example, at least one of polycaprolactone (PCL), ethylcelluloseand polyglycolide or the combination thereof.

In an embodiment of the present disclosure, the using amount of thecarrier is in the range of 300 weight parts to 400 weight parts based onper hundred weight parts of the hydrogen sulfide sustained-releasingmicrospheres.

In an embodiment of the present disclosure, the particle size of thehydrogen sulfide sustained-releasing microspheres is in the range of 8microns to 135 microns.

In an embodiment of the present disclosure, the hydrogen sulfidesustained-releasing microspheres comprise 15 to 25 weight parts of thehydrophobic polymer, 0.5 to 1.5 weight parts of the surfactant and 0.5to 1.5 weight parts of the sodium hydrosulfide.

In an embodiment of the present disclosure, the surfactant can be, forexample, at least one of polysorbate 80, polysorbate 20, polysorbate 60,polysorbate 40, sorbitan oleate, glyceryl oleate and laureth-3, or thecombination thereof.

In an embodiment of the present disclosure, the weight average molecularweight of the hydrophobic polymer can be in the range of 70,000 Da to250,000 Da.

In an embodiment of the present disclosure, the carrier can be, forexample, at least one of Vaseline (petroleum jelly), paraffin gel andbeeswax or the combination thereof.

Another aspect of the present disclosure is to provide a method forpreparing a composition for promoting wound healing. The methodcomprises the steps of: dispersing sodium hydrosulfide in a firstsolvent to form a first solution; adding a hydrophobic polymer into asecond solvent to form a second solution; mixing the first solution andthe second solution to form a third solution; adding the third solutioninto an oil containing a surfactant to form a fourth solution; stirringthe fourth solution; filtering and drying the fourth solution to formhydrogen sulfide sustained-releasing microspheres; heating and melting acarrier; adding the hydrogen sulfide sustained-releasing microspheresinto the melted carrier; and cooling the carrier containing the hydrogensulfide sustained-releasing microspheres.

In an embodiment of the preparing method of the present disclosure, theusing amount of hydrophobic polymer is in the range of 15 to 25 weightparts, the surfactant is in the range of 0.5 to 1.5 weight parts and thesodium hydrosulfide is in the range of 0.5 to 1.5 weight parts.

In an embodiment of the preparing method of the present disclosure, thefirst solvent and the second solvent can be independently at least oneof ethanol, isopropanol, acetone and dimethyl sulfoxide, or thecombination thereof.

In an embodiment of the preparing method of the present disclosure, thehydrophobic polymer can be, for example, at least one ofpolycaprolactone (PCL), ethylcellulose and polyglycolide, or thecombination thereof.

In an embodiment of the preparing method of the present disclosure, theweight average molecular weight of the hydrophobic polymer can be in therange of 70,000 Da to 250,000 Da.

In an embodiment of the preparing method of the present disclosure, thesurfactant can be at least one of polysorbate 80, polysorbate 20,polysorbate 60, polysorbate 40, sorbitan oleate, glyceryl oleate andlaureth-3, or the combination thereof.

In an embodiment of the manufacturing method of the present disclosure,the oil can be at least one of mineral oil, soybean oil, corn oil andsilicon oil or the combination thereof.

In an embodiment of the manufacturing method of the present disclosure,the viscosity of the oil can be in the range of 50 cps to 150 cps.

In an embodiment of the manufacturing method of the present disclosure,the carrier can be, for example, at least one of Vaseline, paraffin geland beeswax or the combination thereof.

In an embodiment of the manufacturing method of the present disclosure,the ratio of the using amount of the hydrogen sulfidesustained-releasing microspheres to the carrier can be in the range of1:3 to 1:4.

The above and other aspects of the present disclosure will become betterunderstood with regard to the following detailed description of thepreferred but non-limiting embodiment(s). These and other aspects of thepresent disclosure will become apparent from the following descriptionof the presently preferred embodiments. The detailed description ismerely illustrative of the present disclosure and does not limit thescope of the present disclosure, which is defined by the appended claimsand equivalents thereof. As would be obvious to one skilled in the art,many variations and modifications of the present disclosure may beaffected without departing from the spirit and scope of the novelconcepts of the present disclosure.

DETAILED DESCRIPTION

In the following detailed description, for purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the disclosed embodiments. It will be apparent,however, that one or more embodiments may be practiced without thesespecific details.

It is apparent that departures from specific designs and methodsdescribed and shown will suggest themselves to those skilled in the artand may be used without departing from the spirit and scope of thepresent disclosure. The present disclosure is not restricted to theparticular constructions described and illustrated, but should beconstrued to cohere with all modifications that may fall within thescope of the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. Generally, thenomenclature used herein and the laboratory procedures are well knownand commonly employed in the art. Conventional methods are used forthese procedures, such as those provided in the art and various generalreferences. Where a term is provided in the singular, the inventors alsocontemplate the plural of that term. The nomenclature used herein andthe laboratory procedures described below are those well-known andcommonly employed in the art.

An aspect of the present disclosure is to provide a novel compositionfor promoting wound healing, which can sustainably release hydrogensulfide, for example, at a releasing concentration in the range of 10 to50 μM/hr in 48 hours. The present composition can prevent from releasinghigh concentration of hydrogen sulfide in a short period of time toinduce cytotoxicity and can effectively promote the healing of chronicwound.

In an embodiment of the composition for promoting wound healing of thepresent disclosure, the composition for promoting wound healing cancomprise a carrier and a plurality of hydrogen sulfidesustained-releasing microspheres. In an embodiment of the presentdisclosure, a ratio of the using amount of the hydrogen sulfidesustained-releasing microspheres and the using amount of the carrier isin the range of 1:3 to 1:4. When the using amount of the carrier isless, the hydrogen sulfide sustained-releasing microspheres cannot besufficiently encapsulated and hydrogen sulfide may be released duringthe preparation process. When the using amount of the carrier isexceeded, the amount of the hydrogen sulfide sustained-releasingmicrospheres will be too less to provide sufficient releasingconcentration of hydrogen sulfide to effectively promote the woundhealing.

In an embodiment of the present disclosure, the carrier is used toencapsulate the hydrogen sulfide sustained-releasing microspheres toelongate the releasing time of the hydrogen sulfide and to enhance thestability of the composition. The suitable carrier can be, but notlimited to, for example, at least one of Vaseline, paraffin gel andbeeswax, or the combination thereof.

In an embodiment of the present disclosure, the hydrogen sulfidesustained-releasing microspheres can comprise a hydrophobic polymer, asurfactant and a sodium hydrosulfide. In an embodiment of the hydrogensulfide sustained-releasing microspheres of the present disclosure, thehydrogen sulfide sustained-releasing microspheres can comprise 15 to 25weight parts of the hydrophobic polymer, 0.5 to 1.5 weight parts of thesurfactant and 0.5 to 1.5 weight parts of the sodium hydrosulfide. Ifthe adding amount of the sodium hydrosulfide is exceeded, the releasedconcentration of the hydrogen sulfide gas is high to cause cytotoxicity.If the adding amount of the sodium hydrosulfide is less, the releasedconcentration of the hydrogen sulfide gas is low to insufficientlypromote the wound healing.

The particle size of the hydrogen sulfide sustained-releasingmicrospheres will affect the releasing rate of the hydrogen sulfide. Inan embodiment of the present disclosure, the suitable particle size ofthe hydrogen sulfide sustained-releasing microspheres can be in therange of 8 microns to 135 microns.

The hydrophobic polymer is used to encapsulate the sodium hydrosulfidefor avoiding the sodium hydrosulfide from contacting moisture to releasehydrogen sulfide gas too quickly, so the releasing rate of the hydrogensulfide can be slowed down and controlled. In an embodiment of thepresent disclosure, the weight average molecular weight of thehydrophobic polymer can be in the range of 70,000 to 250,000. Thesuitable hydrophobic polymer can be at least one of polycaprolactone(PCL), ethylcellulose and polyglycolide, or the combination thereof.

The hydrogen sulfide sustained-releasing microspheres can be prepared byemulsification. The particle sizes, shapes and the surface area of thehydrogen sulfide sustained-releasing microspheres can be adjusted by theratio of the surfactant used with in order to adjust the releasing rateof the hydrogen sulfide. In an embodiment of the present disclosure, thesurfactant can be at least one of polysorbate 80, polysorbate 20,polysorbate 60, polysorbate 40, sorbitan oleate, glyceryl oleate andlaureth-3, or the combination thereof. When the using amount of thesurfactant is exceeded, the particle size of the hydrogen sulfidesustained-releasing microspheres might be too small, the hydrogensulfide sustained-releasing microspheres might have irregular shape orrough surface to cause the releasing rate of the hydrogen sulfide toofast and result in a high releasing concentration thereof. When theusing amount of the surfactant is less, the particle size of thehydrogen sulfide sustained-releasing microspheres might be too large andaffect the releasing rate of the hydrogen sulfide.

The present composition for promoting wound healing is prepared byencapsulating the sodium hydrosulfide by a hydrophobic polymer to form ahydrogen sulfide sustained-releasing microspheres and encapsulating thehydrogen sulfide sustained-releasing microspheres by a carrier.Accordingly, the cytotoxicity caused by high concentration of thehydrogen sulfide can be prevented, the releasing time of the hydrogensulfide can be extended, the stability of the composition can beenhanced, and the users' convenience can be increased.

Another aspect of the present disclosure is to provide a method forpreparing the composition for promoting wound healing. The presentmethod can comprise but not limited to the following steps.

Firstly, sodium hydrosulfide is dispersed into a first solvent to form afirst solution, a hydrophobic polymer is added into a second solvent toform a second solution, and then the first solution and the secondsolution are mixed to form a third solution. In an embodiment of thepreparation method of the present disclosure, when the hydrophobicpolymer is added into the second solvent for preparing the secondsolution, it optionally further comprises a step of heating for meltingthe hydrophobic polymer, the heating temperature can be varied with thehydrophobic polymer used. In a preferred embodiment of the preparationmethod of the present disclosure, the heating temperature can be in therange of 40° C. to 60° C.

In an embodiment of the preparation method of the present disclosure,the using amount of the sodium hydrosulfide can be in the range of 0.5to 1.5 weight parts and the using amount of the hydrophobic polymer canbe in the range of 15 to 25 weight parts.

The first solvent and the second solvent can be the same or different.The suitable first solvent and the suitable second solvent can beindependently at least one of ethanol, isopropanol, acetone and dimethylsulfoxide, or the combination thereof. In an embodiment of thepreparation method of the present disclosure, the first solvent can beethanol and the second solvent can be acetone.

After the third solution is thoroughly stirred, the third solution isadded into an oil containing a surfactant to form a fourth solution, andthen the fourth solution is stirred. In an embodiment of the preparationmethod of the present disclosure, the using amount of the surfactant canbe in the range of 0.5 to 1.5 weight parts.

The suitable surfactant used in the present method can be but notlimited to at least one of polysorbate 80, polysorbate 20, polysorbate60, polysorbate 40, sorbitan oleate, glyceryl oleate and laureth-3, orthe combination thereof.

The particle size of the hydrogen sulfide sustained-releasingmicrospheres can be adjusted by selecting the oil used for and thestirring speed of the fourth solution. In an embodiment of thepreparation method of the present disclosure, the viscosity of the oilcan be in the range of 50 cps to 150 cps. The suitable oil can be butnot limited to, for example, mineral oil, soybean oil, corn oil andsilicon oil, or the combination thereof.

After the fourth solution is stirred, the fourth solution is filteredand dried to form the hydrogen sulfide sustained-releasing microspheres.In an embodiment of the preparation method of the present disclosure,the particle size of the hydrogen sulfide sustained-releasingmicrospheres can be in the range of 8 microns to 135 microns.

Next, the hydrogen sulfide sustained-releasing microspheres are added into a melted carrier. The suitable carrier can be but not limited to, forexample, at least one of Vaseline, paraffin gel and beeswax, or thecombination thereof. In an embodiment of the preparation method of thepresent disclosure, the ratio of the using amount of the hydrogensulfide sustained-releasing microspheres to the using amount of thecarrier can be in the range of 1:3 to 1:4.

Then, the carrier comprising hydrogen sulfide sustained-releasingmicrospheres is cooled down to prepare a composition for promoting woundhealing.

The present disclosure will be described below with reference toExamples to describe the present disclosure in detail but the presentdisclosure is not limited to the description thereof.

EXAMPLE Example 1

0.23 g of sodium hydrosulfide was dispersed in 1.5 ml of ethanol andstirred for 3 hours at room temperature to form a first solution. 2 g ofpolycaprolactone (the weight average molecular weight was about 80,000)was dissolved in 8.5 ml of acetone, and then heated and stirred at 50°C. for 10 minutes to form a second solution. Then, the first solutionand the second solution were mixed and stirred at room temperature for15 minutes to form a third solution. The third solution was dropped into100 ml of mineral oil (Kaydol white mineral oil, commercially obtainedfrom Sonneborn, US) containing 1 ml of sorbitan monooleate (Span80) assurfactant, stirred for 30 minutes at room temperature and filtered thesolution by vacuumed suction to obtain filter cake. The filter cake waswashed by N-heptane and dried in oven for 2 hours to obtain the hydrogensulfide sustained-releasing microspheres. And then, 3.5 g of Vaselinewas heated to 60° C. for liquefying and cooled down to 40° C. 1 g of theresulting microspheres was added into the Vaseline and stirred for 10minutes. The resulting Vaseline mixture was kept stirring until coolingdown to room temperature and being viscous, and then, the mixture wasdried in oven to obtain a composition for promoting wound healing.

Example 2

The steps and materials used in Example 2 are the same as those ofExample 1, except that 0.3 g of sodium hydrosulfide was instead of 0.23g of sodium hydrosulfide, 2 ml of ethanol was instead of 1.5 ml ofethanol and 8 ml of acetone was instead of 8.5 ml of acetone.

Comparative Example 1

The steps and materials used in Comparative example 1 are the same asthose of Example 1, except that 2.5 g of Vaseline was instead of 3.5 gof Vaseline.

Comparative Example 2

The steps and materials used in Comparative example 2 are the same asthose of Example 1, except that 5 g of Vaseline was instead of 3.5 g ofVaseline.

The composition for promoting wound healing obtained from the Examples 1and 2 and Comparative examples 1 and 2 was tested in accordance with thefollowing method.

Determination of the Releasing Rate of the Hydrogen Sulfide

The releasing concentration of the hydrogen sulfide was determined bythe Methylene Blue method. The Methylene Blue method is a known methodfor determining the concentration of sulfide in aqueous solutioncontaining the same in a low level.

Firstly, standard sample with known concentration were prepared forbuilding the calibration curve. The preparation of the calibration curvewas as follows: preparing 0.04 mg/mL sodium hydrosulfide in 500 μLphosphate buffer (pH=7.4) and diluting in series of standard sampleswith different concentration and preparing phosphate buffer withoutsodium hydrosulfide. 100 μL of each of the standard sample was mixedwith 100 μL of 1.0 wt % zinc acetate aqueous solution and then, added 20μL of 20 mM N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N hydrogenchloride aqueous solution and 20 μL of 30 mM FeCl₃ in 1.2 N hydrogenchloride aqueous solution to react 20 minutes at 32° C. The FeCl₃ wasacted as oxidant to catalyze the N,N-dimethyl-p-phenylenediamine sulfateto react with hydrogen sulfide, the product of sodium hydrosulfidedissolved in water, to produce methylene blue. The absorbance of eachstandard sample at wavelength of 670 nm was determined to build thecalibration curve and calculate the concentration of the hydrogensulfide.

Next, each of 10 mg of the compositions for promoting wound healingobtained from Examples 1 and 2 and Comparative examples 1 and 2 wasadded into 1500 μL of phosphate buffer and allowed to stand at 32° C.100 μL of phosphate buffer containing hydrogen sulfide was taken outevery hour and heated at 75° C. for 2 minutes to release the remainedhydrogen sulfide. The phosphate buffer was then added 100 μL of 1.0 wt %zinc acetate aqueous solution, 20 μL of 20 mMN,N-dimethyl-p-phenylenediamine sulfate in 7.2N hydrochloric acid, and20 μL of 30 mM FeCl₃ in 1.2N hydrogen chloride aqueous solution forreacting 20 minutes at 32° C. The resulting solution was detected themethylene blue absorbance at wavelength of 670 nm to determine theaverage released amount of the sodium hydrosulfide of the compositionsobtained from Examples and Comparative examples in every hour during theperiods of 0 to 24 hours and 24 to 48 hours. The test results are shownin the following Table 1.

The Determine Method of the Amount of Sodium Hydrosulfide

Each of 10 mg of the compositions for promoting wound healing obtainedfrom Examples 1 and 2 and Comparative examples 1 and 2 was added into1500 μL of phosphate buffer and were heated at 75° C. for 2 minutes.After hydrogen sulfide was released, 100 μL of phosphate buffercontaining hydrogen sulfide was taken out and added 100 μL of 1.0 wt %zinc acetate aqueous solution, 20 μL of 20 mMN,N-dimethyl-p-phenylenediamine sulfate in 7.2 N hydrogen chlorideaqueous solution, and 20 μL of 30 mM FeCl3 in 1.2 N hydrogen chlorideaqueous solution for reacting 20 minutes at 32° C. The resultingsolution was detected the methylene blue absorbance at wavelength of 670nm to determine the total amount of the sodium hydrosulfide in thecompositions obtained from Examples and Comparative examples. The testresults are shown in the following Table 1.

The test results are shown in Table 1.

TABLE 1 The test results of Examples 1 to 2 and Comparative examples 1to 2 The average The average releasing releasing concentration ofconcentration of The amount hydrogen sulfide hydrogen sulfide of sodiumin 0 to 24 hours in 24 to 48 hours hydrosulfide (μM/hr) (μM/hr) (μM/10mg) Example 1 18.27 10.87 1373.7 Example 2 18.3 14.5 1527.6 Comparative23.55 4.15 840.54 example 1 Comparative 12.72 5.90 649.80 example 2

The composition for promoting wound healing obtained from Examples 1 and2 effectively sustained-released the hydrogen sulfide gas in 48 hourswithout exceeding the safety concentration. The composition forpromoting wound healing obtained from Comparative examples 1 and 2released and maintained at the concentration higher than 10 μM/hr onlyfor 24 hours and less than 10 μM/hr after 24 hours and thus, the woundhealing cannot be effective promoted.

Animal Experiment Result

Animal Experiment were performed using three BKS.Cg-Leprdb/Leprdb/JNarl(leptin receptor-deficient diabetes, db/db, 13 weeks, (commerciallyobtained from National Laboratory Animal Center, Taipei, Taiwan) micewith high plasma glucose levels of >600 mg/dL, which is awell-established diabetic animal model. Mice were anaesthetized withinhaled isoflurane. Three 5-mm-diameter full-thickness skin wounds(volume, approximately 50 μL) were made symmetrically to the dorsal skinof a single mouse with a biopsy punch and treated with the followingdifferent treatments. The wounds of Control group 1 were applied withnormal saline and covered with breathable dressing (Tegaderm™, 3M). Thewounds of Control 2 were applied 20 mg of Vaseline and covered withbreathable dressing. The wounds of Control group 3 were applied 20 mg ofthe composition obtained from Example 2 and covered with breathabledressing. Test mice were housed individually to avoid disturbing woundhealing. The wounds treated with the same manner were in different woundsites on each mouse in order to decrease the factors of wound locationto the wound heal. Wound-closure rates were measured by tracing thewound area every other day.

After the dressings were changed and the wound bed was washed withphosphate buffer solution every other day, the wound area change wasdigitally photographed by Image software. The original wound area as setas 100 and the wound-closure rates was expressed as the percentage ofthe wound area that had healed. The change of the wound area as shown inTable 2.

TABLE 2 the wound healing of Control groups Time (Days) Control group 1Control group 2 Control group 3 0 100 100 100 2  116.9 ± 11.7 117 ± 9 106 ± 7.8  4    106 ± 21.7 101.9 ± 15.4 100.9 ± 7.6  8 48.9 ± 7  48 ± 441.8 ± 14.5 10 22.7 ± 13 17.9 ± 16   0

As shown in Table 2, as time being, the wounds in each control groupwere gradually contracted, but on the tenth day, the wound sites of themice applied with normal saline solution and Vaseline is stillsignificant. The wound sites treated with the composition obtained fromExample 2 were completely healed. Accordingly, the present compositionfor promoting wound healing can effectively promote the wound healing.

Although particular embodiments have been shown and described, it shouldbe understood that the above discussion is not intended to limit thepresent disclosure to these embodiments. Persons skilled in the art willunderstand that various changes and modifications may be made withoutdeparting from the scope of the present disclosure as literally andequivalently covered by the following claims.

What is claimed is:
 1. A composition for promoting wound healingcomprising a carrier; and a plurality of hydrogen sulfidesustained-releasing microspheres, comprising a hydrophobic polymerselected from the group consisting of polycaprolactone (PCL),ethylcellulose, polyglycolide, and combinations thereof; a surfactant;and a sodium hydrosulfide.
 2. The composition as claimed in claim 1,wherein the using amount of the carrier is in the range of 300 weightparts to 400 weight parts based on per hundred weight parts of theplurality of the hydrogen sulfide sustained-releasing microspheres. 3.The composition as claimed in claim 1, wherein the particle size of eachof the plurality of the hydrogen sulfide sustained-releasingmicrospheres is in the range of 8 microns to 135 micron.
 4. Thecomposition as claimed in claim 1, wherein in each of the plurality ofthe hydrogen sulfide sustained-releasing microspheres, the using amountof the hydrophobic polymer is in the range of 15 to 25 weight parts, theusing amount of the surfactant is in the range of 0.5 to 1.5 weightparts, and the using amount of the sodium hydrosulfide is in the rangeof 0.5 to 1.5 weight parts.
 5. The composition as claimed in claim 1,wherein the surfactant is selected from the group consisting ofpolysorbate 80, polysorbate 20, polysorbate 60, polysorbate 40, sorbitanoleate, glyceryl oleate, laureth-3, and combinations thereof.
 6. Thecomposition as claimed in claim 1, wherein the weight average molecularweight of the hydrophobic polymer is in the range of 70,000 Da to250,000 Da.
 7. The composition as claimed in claim 1, wherein thecarrier is selected from the group consisting of petroleum jelly,paraffin gel, beeswax, and combinations thereof.